1. Field of the Invention
The present invention relates to the field of immunoenzymatic assays, also named enzyme immuno-assays.
2. (EIA) Description of the Prior Art
Methods of radio-immunological assay/(RIA) have long been known for the quantitative determination of the concentration of certain substances in biological media, in particular of antigens or of antibodies as well as of certain hormones. However, methods of radio-immunological assay present drawbacks. Among the latter, must be stressed the restrictions in practice which are involved in the use of radioactive labelling agents, which do not permit the method to be accessible to any laboratory indiscriminately.
During the last ten years, important advances accomplished in enzymology have enabled the substitution for radio-immunological assays of immunoenzymatic assays which resort to an enzyme as a labelling agent instead of the radioactive labelling agents previously used. There are numerous bibilographic references in the field of immunoenzymatic assays; it suffices to refer, for example, to the article of S. AVRAMEAS "Detection of Antibodies and Antigens by Means of Enzymes", Bull. Soc. Chim. Biol., 1968, 50, No. 5-6 as well as to the French Patent Applications Nos. 74.34.519 and 75.36.889.
A certain number of immunoenzymatic assay methods exist.
For example, if the method is used for determining antibodies, a method of procedure comprises the steps
of incubating the antibodies to be assayed with an insolubilized antigen,
of washing the solid phase after removal of the supernatant liquors,
of incubating the solid phase with an antibody labelled by means of an enzyme, and
of measuring the enzymatic activity of the solid phase.
In such a method it is possible to utilize any insoluble support on which the antibody is fixed by known means.
In another method called, a "sandwich assay", applied for example, to the determination of antibodies, the following succession of steps is used:
incubating the antibodies to be tested with an antigen rendered insoluble,
washing the solid phase after removal of the supernatant liquors, and
incubating the solid phase with an antigen coupled with an enzyme.
It is also known that the measurements may be carried out by means of the same processes not in the solid phase but in the liquid phase. In all cases, it is the enzymatic activity of the selected phase which is the determinant parameter of the assay.
All the above indications are well-known to the one skilled in the art and have only been given to illustrate the field to which the present invention is applicable.
The method of immunoenzymatic assay has the advantage of being applicable without particular restrictions, which is not the case with radioimmunological assays. However, the practical application of immunoenzymatic assays again raises certain difficulties. In fact, the method is limited as to its sensitivity and does not permit detection of very small amounts of antigen, of antibodies or of hormones in the biological media to be tested. Hence, there exist also cases where, at the present time, no assay method is available if the concentration of the active substance to be measured is too low. It is also important to apply improvements to the methods of coupling between antigens and antibodies with the enzymes, respectively, to improve the fixation yields and to avoid interfering secondary reactions. Finally, problems of stability of the enzyme labelling agents in the course of time may also arise.